Catalog # Aliquot Size

U215-380H-20 20 µg U215-380H-50 50 µg

UBE2D3 (UBCH5C), Active Recombinant full-length human expressed in E. coli cells

Catalog # U215-380H

Lot # V2408-11

Product Description Specific Activity

Recombinant full-length human UBE2D3 was expressed in 840,000 E. coli cells using an N-terminal His tag. The UBE2D3 accession number is NM_003340. 630,000

Gene Aliases 420,000

E2(17)KB3, UBC4/5, UBCH5C 210,000 Activity (RLU) Formulation 0 0 35 70 105 140 Recombinant stored in 50mM sodium phosphate, Protein (ng) pH 7.0, 300mM NaCl, 150mM imidazole, 0.1mM PMSF, 0.25mM DTT, 25% glycerol. The specific activity of UBE2D3 was determined to be 17 nmol /min/mg as per activity assay protocol. Storage and Stability

Purity o Store product at –70 C. For optimal storage, aliquot target into smaller quantities after centrifugation and store at recommended temperature. For most favorable performance, avoid repeated handling and multiple freeze/thaw cycles. The purity of UBE2D3 was determined to be >95% by Scientific Background densitometry, approx. MW 17 kDa.

UBE2D3 also known as UBCH5C or -conjugating enzyme E2D3 is a member of the E2 ubiquitin-conjugating enzyme family which is an important cellular mechanism for targeting abnormal or short-lived proteins for degradation. UBE2D3 is involved in RA-induced cell cycle arrest and as a mediator of all-trans retinoic acid-induced UBE2D3 (UBCH5C), Active cell growth arrest in human acute promyelocytic NB4 Recombinant full-length human protein expressed in E. coli cells cells. UBE2D3 functions in the ubiquitination of the tumor- suppressor protein p53, which is induced by an E3 Catalog # U215-380H ubiquitin-protein ligase and is critical for normal cellular, Specific Activity 17 nmol/min/mg tissue, and organismal physiology. Lot # V2408-11 Purity >95% References Concentration 0.1 µg/µl Stability 1yr at –70oC from date of shipment 1. Mondal, S. et al: A bioluminescent assay for monitoring Storage & Shipping Store product at –70oC. For conjugation of ubiquitin and ubiquitin-like proteins. Anal. optimal storage, aliquot target Biochem. 510: 41-51, 2016 into smaller quantities after 2. Hattori, H. et al: RNAi screen identifies UBE2D3 as a mediator centrifugation and store at of all-trans retinoic acid-induced cell growth arrest in human recommended temperature. For acute promyelocytic NB4 cells. Blood 110: 640-650, 2007. most favorable performance, 3. Hutt, D. et al: The proteome in balance. Science 329: 766- avoid repeated handling and 767, 2010 multiple freeze/thaw cycles. Product shipped on dry ice.

To place your order, please contact us by phone 1-(604)-232-4600, fax 1-604-232-4601 or by email: [email protected] www.signalchem.com

FOR IN VITRO RESEARCH PURPOSES ONLY. NOT INTENDED FOR USE IN HUMAN OR ANIMALS. Activity Assay Protocol

Reaction Components

Active Ubiquitinating Enzymes AMP-GloTM Assay (Promega, Catalog #: V5011) Active UBE2D3 (Catalog #: U215-380H), UBA1 (Catalog AMP, 10 mM #: U201-380G) and BIRC3 (Catalog #: B280-380G) Ultra Pure ATP, 10mM diluted with Ubiquitination Buffer and assayed as outlined AMP-GloTM Reagent I in sample activity plot. (Note: these are suggested AMP-GloTM Reagent II working dilutions and it is recommended that the Kinase-GloTM One Solution researcher perform a serial dilution of Active UBE2D3 for optimal results). Ubiquitination Buffer Substrate (Catalog #: U06-54N)

Buffer components: 40mM Tris (pH7.5), 20mM MgCl2, Wild-type ubiquitin protein diluted with Ubiquitination Buffer 0.1mg/ml BSA. Add 0.5mM DTT prior to use. to a working stock of 170ng/µl (20µM).

Assay Protocol

The UBE2D3 assay is performed using the AMP-GloTM Assay kit (Promega), by detecting the amount of the universal AMP generated. Ubiquitin conjugation is proportional to the generated AMP, and the presence of all components of the Ub conjugation machinery (Ub, E1, E2, and E3) is required for maximal activity of the system. Step 1. Thaw the active UBE2D3, UBA1, BIRC3 and ubiquitin on ice, and all AMP-GloTM components except AMP-GloTM Reagent II at room temperature. Keep AMP-GloTM Reagent II on ice. Step 2. Prepare the following working solutions with Ubiquitination Buffer: o 2X Reaction Cocktail: 170ng/µl ubiquitin + 15ng/µl UBA1 + 40ng/µl BIRC3 + 50µM ATP o 2X final concentration of Active UBE2D3 Step 3. In a half-area white 96-well plate, add the following components to bring the initial reaction volume to 10 µl: Component 1. 5 µl of 2X Reaction Cocktail Component 2. 5 µl of 2X Active UBE2D3 Note: A blank control can be set up as outlined above by replacing the enzyme working solution with an equal volume of Ubiquitination Buffer. Step 4. Briefly centrifuge the plate to ensure reagents are fully mixed and at the bottom of the wells. Seal the plate with a plate seal and incubate at 37°C for 60 minutes Step 5. Equilibrate plate to room temperature. Add 10 µl of AMP-GloTM Reagent I to all wells, mix by shaking for 1-2 minutes. Incubate the plate at room temperature for 60 minutes. Step 6. Prepare AMP Detection Solution by adding AMP-GloTM Reagent II to Kinase-GloTM One Solution at a 1:100 volume ratio. Add 20 µl of the Detection Solution to all wells. Mix for 1-2 minutes and incubate at room temperature for 30 minutes Step 7. Read the plate using the KinaseGlo Luminescence Protocol on a GloMax plate reader (Promega; Cat# E7031) Step 8. Using the AMP standard curve, determine the concentration of AMP produced (µM) and calculate the enzyme specific activity as outlined below. For a detailed protocol of how to determine AMP amount from RLUs, see AMP-Glo™ Assay protocol at Promega’s website: www.promega.com/protocols

Enzyme Specific Activity (SA) (nmol/min/mg)

µ × (µ) = ×10 × ()

To place your order, please contact us by phone 1-(604)-232-4600, fax 1-604-232-4601 or by email: [email protected] www.signalchem.com

FOR IN VITRO RESEARCH PURPOSES ONLY. NOT INTENDED FOR USE IN HUMAN OR ANIMALS.